Efficient Separation of Thermus aquaticus EF-Tu Functional Complexes

tmRNA from Thermus thermophilus

“…Alanyl-tRNA synthetase from T. thermophilus HB8 (M w 195 kDa) with a specific activity of 105 nmolAEmin )1 AEmg )1 (40°C) was obtained generally according to Lechler et al [13]. T. aquaticus EF-Tu (M w 45 kDa) was overproduced in Escherichia coli SCS1 carrying plasmid pTacTU2 with the tufA gene and purified as described in [14]. T7 RNA polymerase was overproduced in E. coli BL21 carrying plasmid pAR1219 and purified as described in [15].…”

“…The protective effect of EF‐Tu against spontaneous hydrolysis of the Ala∼tmRNA or Ala∼tRNA ester bond was studied upon quick dissolution of the dry pellet of purified [ 3 H]Ala∼tmRNA or [ 3 H]Ala∼tRNA in a EF‐Tu*GDPNP‐containing mixture, preincubated for 10 min at the appropriate temperature. The EF‐Tu*GDPNP complex was prepared as described in [14]. [ 3 H]Ala∼tmRNA was synthesized under standard conditions, treated with phenol, separated from low‐molecular‐mass components of the reaction mixture by gel‐filtration on Sephadex G‐25 (Pharmacia) in 50 m m sodium acetate (pH 5.0), and from uncharged tmRNA by chromatography on acetylated DBAE–cellulose (Serva) at 4 °C [17].…”

“…[13]. T. aquaticus EF‐Tu (M w 45 kDa) was overproduced in Escherichia coli SCS1 carrying plasmid pTacTU2 with the tufA gene and purified as described in [14]. T7 RNA polymerase was overproduced in E. coli BL21 carrying plasmid pAR1219 and purified as described in [15].…”

Bacterial elongation factors EF-Tu, their mutants, chimeric forms, and domains: Isolation and purification

“…The hydrophobic chromatography on TSK-Gel Phenyl 5PW with a reversed gradient of ammonium sulfate was shown to efficiently separate the binary complexes of T. thermophilus EF-Tu.GDP, and EF-Tu.GDPNP as well as the ternary complex EF-Tu.GDPNP.Leu-tRNA from each other [82].…”